Recombinant adeno-associated virus delivery of alpha-sarcoglycan polynucleotides

ABSTRACT

The present invention relates to recombinant adeno-associated virus (rAAV) delivery of an alpha-sarcoglycan gene. The invention provides rAAV products and methods of using the rAAV in the treatment of limb girdle muscular dystrophies such as LGMD2D.

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 61/563,139 filed Nov. 23, 2011, whichis incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No.5U54NS055958-03 awarded by the United States National Institute ofHealth/National Institute of Neurological Disorders and Stroke. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to recombinant adeno-associated virus(rAAV) delivery of an alpha-sarcoglycan gene. The invention providesrAAV products and methods of using the rAAV in the treatment of limbgirdle muscular dystrophies such as LGMD2D.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

This application contains, as a separate part of disclosure, a SequenceListing in computer-readable form (filename: 45210PCT_SeqListing.txt;23,573 byte—ASCII text file) which is incorporated by reference hereinin its entirety.

BACKGROUND

Muscular dystrophies (MDs) are a group of genetic diseases. The group ischaracterized by progressive weakness and degeneration of the skeletalmuscles that control movement. Some forms of MD develop in infancy orchildhood, while others may not appear until middle age or later. Thedisorders differ in terms of the distribution and extent of muscleweakness (some forms of MD also affect cardiac muscle), the age ofonset, the rate of progression, and the pattern of inheritance.

One group of MDs is the limb girdle group (LGMD) of MDs. LGMDs are rareconditions and they present differently in different people with respectto age of onset, areas of muscle weakness, heart and respiratoryinvolvement, rate of progression and severity. LGMDs can begin inchildhood, adolescence, young adulthood or even later. Both genders areaffected equally. LGMDs cause weakness in the shoulder and pelvicgirdle, with nearby muscles in the upper legs and arms sometimes alsoweakening with time. Weakness of the legs often appears before that ofthe arms. Facial muscles are usually unaffected. As the conditionprogresses, people can have problems with walking and may need to use awheelchair over time. The involvement of shoulder and arm muscles canlead to difficulty in raising arms over head and in lifting objects. Insome types of LGMD, the heart and breathing muscles may be involved.

There are at least nineteen forms of LGMD, and the forms are classifiedby their associated genetic defects.

Type Pattern of Inheritance Gene or Chromosome LGMD1A Autosomal dominantMyotilin gene LGMD1B Autosomal dominant Lamin A/C gene LGMD1C Autosomaldominant Caveolin gene LGMD1D Autosomal dominant Chromosome 7 LGMD1EAutosomal dominant Chromosome 6 LGMD1F Autosomal dominant Chromosome 7LGMD1G Autosomal dominant Chromosome 4 LGMD2A Autosomal recessiveCalpain-3 gene LGMD2B Autosomal recessive Dysferlin gene LGMD2CAutosomal recessive Gamma-sarcoglycan gene LGMD2D Autosomal recessiveAlpha-sarcoglycan gene LGMD2E Autosomal recessive Beta-sarcoglycan geneLGMD2F Autosomal recessive Delta-sarcoglycan gene LGMD2G Autosomalrecessive Telethonin gene LGMD2H Autosomal recessive TRIM32 LGMD2IAutosomal recessive FKRP gene LGMD2J Autosomal recessive Titin geneLGMD2K Autosomal recessive POMT1 gene LGMD2L Autosomal recessive Fukutingene

Specialized tests for LGMD are now available through a national schemefor diagnosis, the National Commissioning Group (NCG).

U.S. Pat. No. 6,262,035 states it discloses a method for treating apatient suffering from the disease sarcoglycan-deficient limb-girdlemuscular dystrophy by gene replacement therapy. It claims intramuscularinjection of an expression vector containing alpha-sarcoglycan nucleicacid. See also, Allamand et al., Gene Ther., 7(16): 1385-1391 (2000).

The present inventors delivered an alpha-sarcoglycan gene in anadeno-associated type 1 vector by intramuscular injection with the goalof treating LGMD2D as described in Rodino-Klapac et al., Neurology, 71:240-247 (2008); Mendell et al., Ann. Neural., 66(3): 290-297 (2009); andMendell et al., Ann. Neurol., 68(5): 629-638 (2010).

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thesingle-stranded DNA genome of which is about 4.7 kb in length including145 nucleotide inverted terminal repeat (ITRs). There are multipleserotypes of AAV. The nucleotide sequences of the genomes of the AAVserotypes are known. For example, the complete genome of AAV-1 isprovided in GenBank Accession No. NC_002077; the complete genome ofAAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava etal., J. Virol., 45: 555-564 {1983); the complete genome of AAV-3 isprovided in GenBank Accession No. NC_1829; the complete genome of AAV-4is provided in GenBank Accession No. NC_001829; the AAV-5 genome isprovided in GenBank Accession No. AF085716; the complete genome of AAV-6is provided in GenBank Accession No. NC_00 1862; at least portions ofAAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao etal., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided inMol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided inVirology, 330(2): 375-383 (2004). Cis-acting sequences directing viralDNA replication (rep), encapsidation/packaging and host cell chromosomeintegration are contained within the AAV ITRs. Three AAV promoters(named p5, p19, and p40 for their relative map locations) drive theexpression of the two AAV internal open reading frames encoding rep andcap genes. The two rep promoters (p5 and p19), coupled with thedifferential splicing of the single AAV intron (at nucleotides 2107 and2227), result in the production of four rep proteins (rep 78, rep 68,rep 52, and rep 40) from the rep gene. Rep proteins possess multipleenzymatic properties that are ultimately responsible for replicating theviral genome. The cap gene is expressed from the p40 promoter and itencodes the three capsid proteins VP1, VP2, and VP3. Alternativesplicing and non-consensus translational start sites are responsible forthe production of the three related capsid proteins. A single consensuspolyadenylation site is located at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, CurrentTopics in Microbiology and Immunology, 158: 97-129 (1992).

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells, for example, in gene therapy. AAVinfection of cells in culture is noncytopathic, and natural infection ofhumans and other animals is silent and asymptomatic. Moreover, AAVinfects many mammalian cells allowing the possibility of targeting manydifferent tissues in vivo. Moreover, AAV transduces slowly dividing andnon-dividing cells, and can persist essentially for the lifetime ofthose cells as a transcriptionally active nuclear episome(extrachromosomal element). The AAV proviral genome is infectious ascloned DNA in plasmids which makes construction of recombinant genomesfeasible. Furthermore, because the signals directing AAV replication,genome encapsidation and integration are contained within the ITRs ofthe AAV genome, some or all of the internal approximately 4.3 kb of thegenome (encoding replication and structural capsid proteins, rep-cap)may be replaced with foreign DNA. The rep and cap proteins may beprovided in trans. Another significant feature of AAV is that it is anextremely stable and hearty virus. It easily withstands the conditionsused to inactivate adenovirus (56° to 65° C. for several hours), makingcold preservation of AAV less critical. AAV may even be lyophilized.Finally, AAV-infected cells are not resistant to superinfection.

The present inventors have used an AAV8-like AAV termed rh.74 to deliverDNAs encoding various proteins. Xu et al., Neuromuscular Disorders, 17:209-220 (2007) and Martin et al., Am. J. Physiol. Cell. Physiol., 296:476-488 (2009) relate to rh.74 expression of cytotoxic T cell GalNActransferase for Duchenne muscular dystrophy. Rodino-Klapac et al., Mol.Ther., 18(1): 109-117 (2010) describes AAV rh.74 expression of amicro-dystrophin FLAG protein tag fusion after delivery of the AAV rh.74by vascular limb perfusion.

The muscular dystrophies are a group of diseases without identifiabletreatment that gravely impact individuals, families, and communities.The costs are incalculable. Individuals suffer emotional strain andreduced quality of life associated with loss of self-esteem. Extremephysical challenges resulting from loss of limb function createshardships in activities of daily living. Family dynamics suffer throughfinancial loss and challenges to interpersonal relationships. Siblingsof the affected feel estranged, and strife between spouses often leadsto divorce, especially if responsibility for the muscular dystrophy canbe laid at the feet of one of the parental partners. The burden of questto find a cure often becomes a life-long, highly focused effort thatdetracts and challenges every aspect of life. Beyond the family, thecommunity bears a financial burden through the need for added facilitiesto accommodate the handicaps of the muscular dystrophy population inspecial education, special transportation, and costs for recurrenthospitalizations to treat recurrent respiratory tract infections andcardiac complications. Financial responsibilities are shared by stateand federal governmental agencies extending the responsibilities to thetaxpaying community.

There thus remains a need in the art for treatments for musculardystrophies including limb girdle muscular dystrophies such as LGMD2D.

DESCRIPTION

The present invention provides methods and products for preventing,delaying the progression of, and/or treating limb girdle musculardystrophies. The methods involve vascular delivery (e.g., by limbperfusion including, but not limited to, re-circulating metholodogy) ofan alpha-sarcoglycan expression cassette to muscle cells using AAV as agene delivery vector. For example, the alpha sarcoglycan expressioncassette is inserted in the genome of the AAV referred to as AAV rh.74herein.

In one aspect, the invention provides an AAV referred to as AAV rh.74.AAV rh.74 exhibits about 93% identity to AAV8 capsid. FIG. 1 provides analignment of the AAV rh.74 capsid amino acid sequence with the AAV8capsid amino acid sequence. The polynucleotide and amino acid sequencesof the AAV rh.74 capsid are respectively set out in SEQ ID NOs: 1 and 2.

In another aspect, a method of ameliorating limb girdle musculardystrophy type 2D (LGMD) in a patient is provided. In some embodiments,the method comprises the step of perfusing the vasculature of a limb ofthe patient with a rAAV comprising the AAV rh.74 capsid of SEQ ID NO: 2and comprising an alpha-sarcoglycan polynucleotide (for example, thepolynucleotide of SEQ ID NO: 3) in a gene expression cassette in thevirus genome.

In yet another aspect, the invention provides a method of inhibiting theprogression of dystrophic pathology associated with LGMD 2D. In someembodiments, the method comprises the step of perfusing the vasculatureof a limb of the patient with a rAAV comprising AAV rh.74 capsid of SEQID NO: 2 and comprising an alpha-sarcoglycan polynucleotide (forexample, the polynucleotide of SEQ ID NO: 3) in a gene expressioncassette in the virus genome.

In still another aspect, a method of improving muscle function in apatient afflicted with limb girdle muscular dystrophy type 2D (LGMD2D)is provided. In some embodiments, the method comprises the step ofperfusing the vasculature of a limb of the patient with a rAAVcomprising AAV rh.74 capsid of SEQ ID NO: 2 and comprising analpha-sarcoglycan polynucleotide (for example, the polynucleotide of SEQID NO: 3) in a gene expression cassette in the virus genome. In someinstances, the improvement in muscle function is an improvement inmuscle strength. The improvement in muscle strength is determined bytechniques known in the art such as the maximal voluntary isometriccontraction testing (MVICT). In some instances, the improvement inmuscle function is an improvement in stability in standing and walking.The improvement in stability strength is determined by techniques knownin the art such as the 6-minute walk test (6MWT) or timed stair climb.

In another aspect, the invention provides a method of delivering analpha-sarcoglycan polynucleotide to an animal (including, but notlimited to, a human). In some embodiments, the method comprises the stepof perfusing the vasculature of a limb of the animal with a rAAVcomprising the AAV rh.74 capsid of SEQ ID NO: 2 and comprising an alphasarcoglycan polynucleotide (for example, the polynucleotide of SEQ IDNO: 3) in a gene expression cassette in the virus genome.

Cell transduction efficiencies of the methods of the invention describedabove and below may be at least about 60, 65, 70, 75, 80, 85, 90 or 95percent. In some embodiments, transduction efficiency is increased byincreasing the volume of the composition in which the rAAV is delivered,pre-flushing before delivery of the rAAV and/or increasing dwell time ofthe rAAV.

In some embodiments of the foregoing methods of the invention, the virusgenome is a self-complementary genome. In some embodiments of themethods, the genome of the rAAV lacks AAV rep and cap DNA. In someembodiments of the methods, the rAAV is AAVrh.74.tMCK.hSGCA.

In yet another aspect, the invention provides a rAAV comprising the AAVrh.74 capsid of SEQ ID NO: 2 and comprising an alpha sarcoglycanpolynucleotide (for example, the polynucleotide of SEQ ID NO: 3) in agene expression cassette in the virus genome. In some embodiments, thegenome of the rAAV lacks AAV rep and cap DNA. In some embodiments, therAAV is a self-complementary genome. In some embodiments, the rAAV isAAVrh.74.tMCK.hSGCA.

Recombinant AAV genomes of the invention comprise one or more AAV ITRsflanking a polynucleotide encoding alpha sarcoglycan. The polynucleotideis operatively linked to transcriptional control DNA, specificallypromoter and polyadenylation signal DNAs that are functional in target,forming an expression cassette. AAV DNA in the rAAV genomes may be fromany AAV serotype for which a recombinant virus can be derived including,but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11. As noted in theBackground section above, the nucleotide sequences of the genomes ofvarious AAV serotypes are known in the art. In some embodiments of theinvention, the promoter DNAs are muscle-specific control elements,including, but not limited to, those derived from the actin and myosingene families, such as from the myoD gene family [See Weintraub et al.,Science, 251: 761-766 (1991)], the myocyte-specific enhancer bindingfactor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11: 4854-4862(1991)], control elements derived from the human skeletal actin gene[Muscat et al., Mol. Cell. Biol., 7: 4089-4099 (1987)], the cardiacactin gene, muscle creatine kinase sequence elements [Johnson et al.,Mol. Cell. Biol., 9:3393-3399 (1989)] and the murine creatine kinaseenhancer (MCK) element, desmin promoter, control elements derived fromthe skeletal fast-twitch troponin C gene, the slow-twitch cardiactroponin C gene and the slow-twitch troponin I gene: hypozia-induciblenuclear factors [Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)], steroid-inducible elements and promoters includingthe glucocorticoid response element (GRE) [See Mader and White, Proc.Natl. Acad. Sci. USA, 90: 5603-5607 (1993)], and other control elements.

DNA plasmids of the invention comprise rAAV genomes of the invention.The DNA plasmids are transferred to cells permissible for infection witha helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus orherpesvirus) for assembly of the rAAV genome into infectious viralparticles. Techniques to produce rAAV particles, in which an AAV genometo be packaged, rep and cap genes, and helper virus functions areprovided to a cell are standard in the art. Production of rAAV requiresthat the following components are present within a single cell (denotedherein as a packaging cell): a rAAV genome, AAV rep and cap genesseparate from (i.e., not in) the rAAV genome, and helper virusfunctions. The AAV rep genes may be from any AAV serotype for whichrecombinant virus can be derived and may be from a different AAVserotype than the rAAV genome ITRs, including, but not limited to, AAVserotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9,AAV-10 and AAV-11. Use of cognate components is specificallycontemplated. Production of pseudotyped rAAV is disclosed in, forexample, WO 01/83692 which is incorporated by reference herein in itsentirety.

A method of generating a packaging cell is to create a cell line thatstably expresses all the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line is then infected with a helpervirus such as adenovirus. The advantages of this method are that thecells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus rather than plasmids to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658.776 ;WO 95/13392; WO 96/17947; PCT/U598/18600; WO 97/09441 (PCT/US96/14423);WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. No. 5,786,211; U.S. Pat.No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing documents arehereby incorporated by reference in their entirety herein, withparticular emphasis on those sections of the documents relating to rAAVproduction.

The invention thus provides packaging cells that produce infectiousrAAV. In one embodiment packaging cells may be stably transformed cancercells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293line). In another embodiment, packaging cells are cells that are nottransformed cancer cells, such as low passage 293 cells (human fetalkidney cells transformed with E1 of adenovirus), MRC-5 cells (humanfetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells(monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

The rAAV may be purified by methods standard in the art such as bycolumn chromatography or cesium chloride gradients. Methods forpurifying rAAV vectors from helper virus are known in the art andinclude methods disclosed in, for example, Clark et al., Hum. GeneTher., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.

In another embodiment, the invention contemplates compositionscomprising rAAV of the present invention. Compositions of the inventioncomprise rAAV in a pharmaceutically acceptable carrier. The compositionsmay also comprise other ingredients such as diluents. Acceptablecarriers and diluents are nontoxic to recipients and are preferablyinert at the dosages and concentrations employed, and include bufferssuch as phosphate, citrate, or other organic acids; antioxidants such asascorbic acid; low molecular weight polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, pluronics or polyethylene glycol (PEG).

Titers of rAAV to be administered in methods of the invention will varydepending, for example, on the particular rAAV, the mode ofadministration, the treatment goal, the individual, and the cell type(s)being targeted, and may be determined by methods standard in the art.Titers of rAAV may range from about 1×10⁶, about 1×10⁷, about 1×10⁸,about 1×10⁹, about 1×10¹⁰, about 1×10¹¹, about 1×10¹², about 1×10¹³ toabout 1×10¹⁴ or more DNase resistant particles (DRP) per ml. Dosages mayalso be expressed in units of viral genomes (vg) (Le., 1×10⁷ vg, 1×10⁸vg, 1×10⁹vg, 1×10¹⁰ vg, 1×10¹¹ vg, 1×10¹² vg, 1×10¹³ vg, 1×10¹⁴ vg,respectively).

Methods of transducing a target cell (e.g., a skeletal muscle, smoothmuscle or cardiac muscle cell) with rAAV, in vivo or in vitro, arecontemplated by the invention. The methods comprise the step ofadministering an effective dose, or effective multiple doses, of acomposition comprising a rAAV of the invention to an animal (including ahuman being) in need thereof. If the dose is administered prior todevelopment of a LGMD2D, the administration is prophylactic. If the doseis administered after the development of LGMD2D, the administration istherapeutic. In embodiments of the invention, an effective dose is adose that alleviates (eliminates or reduces) at least one symptomassociated with LGMD2D being treated, that slows or prevents progressionto LGMD2D, that slows or prevents progression of a disorder/diseasestate, that diminishes the extent of disease, that results in remission(partial or total) of disease, and/or that prolongs survival.

Combination therapies are also contemplated by the invention.Combination as used herein includes simultaneous treatment or sequentialtreatments. Combinations of methods of the invention with standardmedical treatments (e.g., corticosteroids and/or immunosuppressivedrugs) are specifically contemplated, as are combinations with noveltherapies.

Sterile injectable solutions are prepared by incorporating rAAV in therequired amount in the appropriate solvent with various otheringredients enumerated above, as required, followed by filtersterilization. Generally, dispersions are prepared by incorporating thesterilized active ingredient into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying technique that yield a powder of theactive ingredient plus any additional desired ingredient from thepreviously sterile-filtered solution thereof.

The composition comprising a rAAV of the invention may also beadministered to an animal (including a human being) in need thereofusing a system such as is illustrated in FIG. 5, and/or according to amethod such as illustrated in FIG. 6. In this regard, FIGS. 7 and 8 mayhave been simplified by the omission of selected elements for thepurpose of more clearly showing other elements. Such omissions ofelements in some figures are not necessarily indicative of the presenceor absence of particular elements in any of the exemplary embodiments,except as may be explicitly delineated in the corresponding writtendescription. FIG. 5 is not necessarily to scale.

FIG. 5 illustrates an exemplary system 100 that may be used according tothe present disclosure to deliver the rAAV, potentially in combinationwith other treatments and therapies, according to an isolated whole limbrecirculation protocol. The system 100 includes a venous catheter 102, apump 104, an optional oxygenator 106, an optional heat exchanger 108(which may according to certain embodiments be formed integrally withthe oxygenator 106), and an arterial catheter 110. In addition, thesystem 100 may include a one or more sets defining a circuit 120connecting the venous catheter 102 to the arterial catheter 110, andreceived within or connected to the pump 104, the oxygenator 106, andthe heat exchanger 108. These sets may include one or more connectors,which may be luer-type connectors, as well as tubing and reservoirs.

In fact, according to one embodiment of the present circuit 120, thecircuit 120 may include a first connection (and collection) site 122 anda second connection (and introduction) site 124. Either or both of theconnection sites 122, 124 may be defined by a luer connectorincorporating a stopcock, permitting fluid to be diverted from thecircuit 120. For example, the first connection site 122 may includefirst and second luer connectors, each with a stopcock and attachedline. The second connection site 124 may include a single stopcock withattached line. The lines running between the catheters 102, 110, theother equipment (pump 104, optional oxygenator 106, optional heatexchanger 108) and the sites 122, 124 may be exaggerated in FIG. 5 forease of illustration.

As illustrated, the venous catheter 102 is connected to a first end ofthe circuit 120 that is received in the pump 104, which may be aperistaltic or roller pump according to certain embodiments. The circuit120 may also be connected to the oxygenator 106 if the perfusate passingthrough the circuit 120 is blood, for example. If fluids other thanblood are passed through the circuit 120 between the catheters 102, 110,then the oxygenator 106 may not be required. Additionally, the circuit120 may be received or connected to a heat exchanger 108, which heatexchanger 108 may be used to control or maintain the temperature of thefluid passing through the circuit 120. As noted above, the heatexchanger 108 is presently believed to be optional, and may not beincluded in all embodiments of the present disclosure. The circuit 120is connected at a second end to the arterial catheter 110.

The system 100 may be connected to a patient 150, and in particular, toa limb 152 (e.g., lower extremity) of the patient 150 that has beenisolated from the remainder of the patient's body 154 by a tight ortightly-placed tourniquet 156, which is provided as an exemplaryisolation device or system. The venous catheter 102 may be placed ordisposed within a vein 160 (e.g., femoral vein) of the limb 152, whilethe arterial catheter 110 may be placed or disposed within an artery 162(e.g., femoral artery) of the limb 152. As illustrated, the insertionsite of the venous catheter 102 and the insertion site of the arterialcatheter 110 are adjacent each other and only slightly distal of thetourniquet 156.

Where the limb 152 is the lower extremity (i.e., a leg), both cathetersites may be in the groin region. However, according to otherembodiments, the venous catheter 102 may be disposed only slightlydistal to the tourniquet 156, while the arterial catheter 110 isdisposed at a considerable distance from the tourniquet 156. Forexample, where the limb 152 is a leg, one site may be in the groin, andthe other at the ankle. According to still other embodiments, an upperextremity (i.e., an arm) may be targeted.

The catheters 102, 110 may be introduced into the vein and artery,respectively, either by surgical cut down and blunt dissection or byless invasive procedures, such as the Seldinger technique(percutaneously). In regard to the later technique, it may be possibleto introduce the catheters at a location remote to the limb undergoingperfusion and to advance the catheters from the remote site to alocation proximate to the limb to be perfused. In fact, ballooncatheters may be used to perform both the connection to the circuit 120and (when inflated) the isolation of the limb from the remainder thepatient's body.

The system 100 is operated to circulate a fluid, which may be referredto as the perfusate, from the point of insertion of the arterialcatheter 110 through the limb 152 to the point of insertion of thevenous catheter 110, through the pump 104, optional oxygenator 106, andoptional heat exchanger 108, and back to the arterial catheter 110. Asnoted above, certain embodiments may employ the patient's blood,potentially in combination with additional blood or blood components.However, according to certain embodiments of the present disclosure, theperfusate may be saline or a buffer solution. According to a non-bloodperfusate embodiment, the blood may be removed from the limb 152 via thevenous catheter 102 (and the site 122), while the perfusate isintroduced via the arterial catheter 110 (and the site 124).

The system 100 may also include sensors that may be used to monitor theflow of the perfusate through the system 100 and the limb 152, and mayeven be used to control the operation of the pump 104, for example. Inparticular, pressure sensors 170, 172 may be disposed upstream (venousside) and downstream (arterial side) of the pump 104 and optionaloxygenator 106 and heat exchanger 108. In particular, the sensor 170 maybe used to determine if a low pressure condition is occurring onupstream of the pump 104 such that the operation of the pump 104 shouldbe stopped momentarily to prevent damage to the blood vessels of thelimb 152.

A method 200 of operating the system 100 is illustrated in FIG. 6 todeliver an rAAV. It will be understood that the method 200 may becarried out using equipment other than that illustrated in FIG. 5 (i.e.,system 100). In addition, it will be understood that the system 100 maybe used to carry out a method other than the method 200 illustrated inFIG. 6. However, according to certain embodiments, the system 100 may beoperated in accordance with the method 200.

The method 200 begins at block 202 with the insertion of the catheters102, 110, or at least with the insertion of the venous catheter 102 intothe vein 160. At block 202, the circuit 120 may also be connected to thecatheters 102, 110 (and thereby to the vasculature of the limb 152).Method 200 continues at block 204 with the isolation of the limb 152from the remainder 154 of the body, which may be achieved by applyingthe tourniquet 156 to the limb 152 for example. It will be recognizedthat the order of the steps of blocks 202 and 204 may in fact bereversed according to certain embodiments of the present method.Depending on the choice of perfusate, the method 200 may then proceed tooptional block 206.

If the perfusate is other than blood (i.e., a non-blood perfusate), thenat block 206 a volume of the patient's blood is removed from the limb152 via the venous catheter 102 and the site 122 while the non-bloodperfusate (e.g., a buffer solution, such as Normosol-R available fromHospira Inc., Lake Forest, Ill.) is introduced into the circuit 120 andthe limb 152 via the site 124 and the arterial catheter 110. The bloodmay be disposed in a sterile blood bag, and an anti-coagulant may beadded to the blood, such as ACD-A or Heparin, for storage. The volume ofblood would then be stored using conventional methods for laterreintroduction, as explained in detail below.

Alternatively, the patient's own blood may be used as the perfusate.However, if the patient's own blood is used, then it may be advisable toprovide for oxygenation of the blood by way of the optional oxygenator106. Moreover, it may also be advisable to screen the blood forantibodies and complements that have specific binding sites for therAAV, or that exhibit non-specific biding with the rAAV. If the patientis naïve to the rAAV, no further action may be required. However, if thepatient has antibodies or complements that exhibit specific ornon-specific binding with the rAAV, the blood may need to be filteredbefore it is used as the perfusate in the method 200. For example,plasmapheresis may be used to remove the antibodies and/or complementsfrom the patient's blood.

In either event, some additional perfusate may be added to facilitatethe travel of the rAAV within the limb 152, and in particular within themuscles of the limb 152. However, because vascular pressures that areexcessive are believed to be detrimental to the tissue of the limb 152,overall volume of perfusate used is minimized according to certainembodiments of the present disclosure.

Once the step of block 206 has been performed, if required, the pump 104is activated to cause the perfusate to circulate through the limb 152 toperfuse limb 152 at block 208. According to certain embodiments of thepresent disclosure, after perfusion of the limb 152 has begun, the rAAVmay be administered at block 210 through its introduction (injection)into the circuit 120 and the limb 152, for example via the arterialcatheter 110. According to other embodiments, the administration of therAAV may occur prior to the activation of the pump 104 or may be delayedsome period of time after the perfusion has begun. The rAAV is thenpermitted to recirculate, or pass repeatedly, with the perfusate throughthe circuit 120 and limb 152 for a period of time at block 212.

The period of time that the perfusate and rAAV is recirculating may bevaried from patient to patient, and between treatments of the samepatient. For example, the time period may be at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 minutes. For that matter, the time period may be at least10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 1 hour.According to certain embodiments, the time period may even exceed 3hours, although many treatments may be less than 2 hours in length.According to particular embodiments, the time period may beapproximately or about 30 minutes.

Once the recirculation of the perfusate and rAAV has been conducted overthe desired time period, the perfusate and any rAAV remaining in theperfusate may be removed from the circuit 120 and the limb 152 at block214. While it may be possible to perform a flush of the limb 152 byintroducing a perfusate without rAAV as the perfusate with residual rAAVis removed, it is not expected that such a flush will be routinelyperformed. It also may be possible to reintroduce or reperfuse the limb152 at this point with the volume of the patient's blood removed fromthe limb 152 at the beginning of the procedure, although this also maynot occur according to all embodiments of the present method 200.

Once the perfusate has been replaced with the blood at block 214 (ifdesired), the isolation of the limb 152 may be discontinued, by removingthe tourniquet 156 for example, at block 216. At this point orimmediately prior to block 216, the catheters 102, 110 may be removedfrom the vein 160 and artery 162 of the limb 152 at block 218, therebydisconnecting the circuit 120 from the limb 152.

It is believed that the use of a recirculating system, such as thesystem 100, and a method of recirculation, such as the method 200, mayhave one or more advantages with regard to the administration of therAAV. To begin, the recirculation thus described facilitates the travelof the rAAV to all or nearly all regions of the limb 152, and inparticular to all or nearly all of the muscle fibers of the muscles ofthe limb 152, and for those regions to be exposed to the rAAV multipletimes. Both the widespread nature of the exposure, as well as theduration/frequency of the exposure, are believed to assist in the rAAVtransferring the genetic material into the muscle cells and theinterstitial spaces between the muscle fiber cells of the targeted limb152. However, further advantages may be obtained when a non-oxygenatedperfusate is used in the system 100 and the limb 152. It will berecognized that if a non-oxygenated perfusate (e.g., a buffer solution)is used in the system 100 and limb 152, the tissue of the limb willexperience hypoxia and/or acidosis over time because of the lack ofoxygen in the circulating perfusate. Hypoxia and acidosis are known tocause blood vessels to dilate (vasodilatation). As a consequence, it isbelieved that the travel of the perfusate and the rAAV carried by theperfusate will be further facilitated because of the dilated nature ofthe vessels, permitting the perfusate and rAAV to travel deep within thetissues of the targeted limb 152.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an alignment of the AAV rh.74 (SEQ ID NO: 2) and AAVB capsid(SEQ ID NO: 4) amino acid sequences.

FIG. 2 shows the tMCK-aSG gene cassette.

FIG. 3 shows the sc.tMCk.aSG vector plasmid.

FIG. 4 is the rh74 genome sequence (SEQ ID NO: 5) wherein nucleotides210-2147 are the Rep 78 gene open reading frame, 882-208 are the Rep52open reading frame, 2079-2081 are the Rep78 stop, 2145-2147 are theRep78 stop, 1797-1800 are a splice donor site, 2094-2097 are a spliceacceptor site, 2121-2124 are a splice acceptor site, 174-181 are the p5promoter +1 predicted, 145-151 are the p5 TATA box, 758-761 are the p19promoter +1 predicted, 732-738 are the p19 TATA box, 1711-1716 are thep40 TATA box, 2098-4314 are the VP1 Cap gene open reading frame,2509-2511 are the VP2 start, 2707-2709 are the VP3 start and 4328-4333are a polyA signal.

FIG. 5 is a schematic illustration of a system for recirculating therAAV according to the present disclosure.

FIG. 6 is a flowchart of a method of recirculating rAAV according to thepresent disclosure.

FIG. 7 shows the average transgene expression throughout the lowerextremity following vascular delivery and recirculation of 6×10¹² vg/kgof rAAV comprising an enhanced green fluorescent protein (eGFP)transgene, AAVrh.74.CMD.eGFP. A) Each representative panel is a directfluorescent image of a section of muscle demonstrating the extent ofeGFP expression. The Biceps (a non-targeted muscle from the upperextremity is presented as a negative control). B) Each bar represents anaverage of two muscles—one from each lower extremity of a Rhesus macaqueand shows the percent muscle fiber transgene expression of the majorlower extremity muscles. QVL=Vastus Lateralis, QRF=Rectus Femoris,QVM=Vastus Medialis, QVI=Vastus Intermedius, HBF=Biceps Femoris,HSM=Semimembranosus, HST=Semitendinosus, HSart=Sartorius,HGrac=Gracilis, GlutMax=Gluteus Max, GlutMed=Gluteus Med, TA=TibialisAnterior, EDL=Extensor Digitorum Longus, MG=Medial Gastrocnemius,LG=Lateral Gastrocnemius, Sol=Soleus.

FIG. 8 shows the average transgene expression throughout the lowerextremity following vascular delivery and recirculation of 2×10¹² vg/kgof vector (AAVrh.74.CMD.eGFP). A) Each representative panel is a directfluorescent image of a section of muscle demonstrating the extent oftransgene expression. The Biceps (a non-targeted muscle from the upperextremity is presented as a negative control). B) Each bar represents anaverage of two muscles—one from each lower extremity of a Rhesus macaqueand shows the percent muscle fiber transgene expression of the majorlower extremity muscles. QVL=Vastus Lateralis, QRF=Rectus Femoris,QVM=Vastus Medialis, QVI=Vastus Intermedius, HBF=Biceps Femoris,HSM=Semimembranosus, HST=Semitendinosus, HSart=Sartorius,HGrac=Gracilis, GlutMax=Gluteus Max, GlutMed=Gluteus Med, TA=TibialisAnterior, EDL=Extensor Digitorum Longus, MG=Medial Gastrocnemius,LG=Lateral Gastrocnemius, Sol=Soleus.

FIG. 9 shows the average transgene expression throughout the lowerextremity following vascular delivery and recirculation of 6×10¹² vg/kgof AAVrh.74.MCK.micro-dystrophin. A) Each representative panel is animmunofluorescent image of a section of muscle demonstrating the extentof transgene expression. The Biceps (a non-targeted muscle from theupper extremity is presented as a negative control). B) Each barrepresents an average of two muscles—one from each lower extremity of aRhesus macaque and shows the percent muscle fiber transgene expressionof the major lower extremity muscles. QVL=Vastus Lateralis, QRF=RectusFemoris, QVM=Vastus Medialis, QVI=Vastus Intermedius, HBF=BicepsFemoris, HSM=Semimembranosus, HST=Semitendinosus, HSart=Sartorius,HGrac=Gracilis, GlutMax=Gluteus Max, GlutMed=Gluteus Med, LCF=LateralCaudal Femoris, TA=Tibialis Anterior, EDL=Extensor Digitorum Longus,MG=Medial Gastrocnemius, LG=Lateral Gastrocnemius, Sol=Soleus.

FIG. 10 shows the average transgene expression throughout the lowerextremity following vascular delivery and recirculation of 6×10¹² vg/kgof AAVrh.74.tMCK.SGCA. A) Each representative panel is animmunofluorescent image of a section of muscle demonstrating the extentof transgene expression. The Biceps (a non-targeted muscle from theupper extremity is presented as a negative control). B) Each barrepresents the average of two muscles—one from each lower extremity of aRhesus macaque and shows the percent muscle fiber transgene expressionof the major lower extremity muscles. QVL=Vastus Lateralis, QRF=RectusFemoris, QVM=Vastus Medialis, QVI=Vastus Intermedius, HBF=BicepsFemoris, HSM=Semimembranosus, HST=Semitendinosus, HSart=Sartorius,HGrac=Gracilis, GlutMax=Gluteus Max, GlutMed=Gluteus Med, LCF=LateralCaudal Femoris, TA=Tibialis Anterior, EDL=Extensor Digitorum Longus,MG=Medial Gastrocnemius, LG=Lateral Gastrocnemius, Sol=Soleus.

FIG. 11 shows rAAV.rh.74.tMCK.SGCA gene transfer restores specific forceand resistance to eccentric contractions in the EDL of alpha-sarcoglycanknock-out mice. Alpha-sarcoglycan knock-out mice (n=12 per group) weretreated by ILP at high (2×10¹² vg/kg) and low (6×10¹¹ vg/kg) doses.

EXAMPLES

Thus, aspects and embodiments of the invention are illustrated by thefollowing examples. Example 1 describes the isolation of AAV rh.74.Example 2 describes alpha-sarcoglycan gene expression from a highlyactive expression cassette combined with a self-complementary AAVvector. Example 3 describes gene delivery via the mouse vasculatureusing AAV rh.74. Example 4 describes the vascular delivery ofAAVrh.74.tMCK.hSGCA in non-human primates. Example 5 describes thebiodistribution of the AAVrh.74.tMCK.hSGCA vector in the macaques.Example 6 describes administration of AAVrh.74.tMCK.hSGCA to a humanpatient. Example 7 describes isolated whole limb re-circulation (IWRLC)methodology according to the invention. Example 8 describes IWLRC in thenon-human primate with a reporter construct. Example 9 describes IWLRCin the non-human primate with therapeutic transgenes. Example 10describes vascular delivery of SC rAAV8.tMCK.hSGCA to alpha-sarcoglycanknock-out mice.

Example 1 Isolation of AAV rh.74

A unique AAV serotype was isolated from a rhesus macaque lymph nodeusing a novel technique termed Linear Rolling Circle Amplification.Using the LRCA process, double-stranded circular AAV genomes wereamplified from several rhesus macaques. The method is predicated on theability to amplify circular AAV genomes by isothermic rolling circleamplification using phi29 phage DNA polymerase and AAV specific primers.LRCA products are contiguous head-to-tail arrays of the circular AAVgenomes from which full-length AAV Rep-Cap molecular clones wereisolated. Four isolates were sequenced and the predicted amino acidsequences for Rep and Cap ORFs were aligned and compared to previouslypublished serotypes (Table). VP1 protein sequences were analyzed andrevealed homology to the NHP AAV clades D, E, and AAV 4-like virusisolates. Analysis of the Rep78 (top portion of Table) ORF revealedstrong homology to AAV 1 (98-99%).

One macaque tissue sample (rh426-M) yielded a divergent AAV8-likeisolate termed rh.74 that shares 93% sequence identity with AAV8. Thenucleotide and amino acid sequences of the rh.74 capsid gene arerespectively set out in SEQ ID NOs: 1 and 2. FIG. 1 shows an alignmentof the rh.74 (SEQ ID NO: 2) and the AAV8 capsid (SEQ ID NO: 4) aminoacid sequences.

The rh.74 capsid gene sequence was cloned into an AAV helper plasmidcontaining the Rep gene from AAV2 to provide vector replicationfunctions for recombinant AAV vector production.

Example 2 Robust Alpha-Sarcoglycan Gene Expression Using a Highly ActiveExpression Cassette Combined with a Self-Complementary AAV Vector

A vector was designed with several features to maximize the opportunityfor clinical success. First, to ameliorate possible immune responses tothe vector expression cassette, a synthetic codon-optimized humanalpha-sarcoglycan cDNA (hSCGA) was placed under the control of a musclespecific promoter (the truncated muscle creatine kinasepromoter/enhancer). The tMCK promoter was a gift from Dr. Xiao Xiao(University of North Carolina). It is a modification of the previouslydescribed CK6 promoter [Shield et al., Mol Cell Biol, 16:5058-5068(1996)] and includes a modification in the enhancer upstream of thepromoter region containing transcription factor binding sites. Theenhancer is composed of two E-boxes (right and left). The tMCK promotermodification includes a mutation converting the left E-box to a rightE-box (2R modification) and a 6 bp insertion (S5 modification). Thenucleotide sequence of the hSCGA is set out in SEQ ID NO: 3. Second, theconstruct also includes a chimeric intron to promote high levelexpression. The chimeric intron is composed of the 5′ donor site fromthe first intron of the human (3-globin gene and the branchpoint and 3′splice acceptor site from the intron that is between the leader and thebody of an immunoglobulin gene heavy chain variable region. Third, asynthetic SV40 polyadenylation signal is used for efficienttranscription termination. A schematic of the expression cassette isshown below in FIG. 2.

The expression cassette was inserted into the pHpa7 self-complementaryAAV vector plasmid backbone to generate plasmid sc.tMCK.aSG shown inFIG. 3. The location of the expression cassette elements in the plasmidis given in Table 2 below.

TABLE 2 Type Start End Name Description REGION 1 116 ITR Invertedterminal repeat REGION 147 860 tMCKp Truncated MCK promoter REGION 8911024 sd/sa Chimeric intron GENE 1064 2228 ha-SG Human alpha sarcoglycangene REGION 2229 2280 pA SV40 late polyadenylation signal REGION 23772480 ITR Inverted terminal repeat

To maximize vector potency and reduce the dosing requirements, aself-complementary (SC) AAV vector was produced. SC AAV vectorsdemonstrate increased gene expression and express the protein productsooner than standard single-stranded AAV vectors. This improvement isachieved by deleting a small portion of one AAV inverted terminal repeat(ITR) that causes AAV replication to proceed to a dimeric replicationintermediate that is then packaged into AAV particles.

The recombinant SC AAV vector (AAVrh.74.tMCK.hSGCA) expressing thealpha-sarcoglycan gene from the muscle specific tMCK promoter wasproduced by a modified cross-packaging approach using the plasmidsc.tMCK.aSG in an adenovirus-free, triple plasmid DNA transfection(CaPO₄ precipitation) method in HEK293 cells [Rabinowitz et al., J.Virol., 76:791-801 (2002)]. Vector was produced by co-transfecting withan AAV helper plasmid rep2-cap rh.74 and an adenovirus helper plasmid insimilar fashion as that previously described [Wang et aL, Gene. Ther.,10:1528-1534 (2003)]. Plasmid rep2-cap rh.74 encodes the wild-type AAV2rep gene and rh.74 cap gene, and the adenovirus helper plasmid(pAdhelper) expresses the adenovirus type 5 E2A, E4ORF6, and VA I/II RNAgenes which are required for high-titer rAAV production.

Vectors were purified from clarified 293 cell lysates by sequentialiodixanol gradient purification and anion-exchange column chromatographyusing a linear NaCl salt gradient as previously described [Clark et al.,Hum. Gene Ther, 10:1031-1039 (1999)]. Vector genome (vg) titers weremeasured using QPCR based detection with a tMCK specific primer/probeset and utilized the Prism 7500 Taqman detector system (PE AppliedBiosystems) as previously described (Clark et al., supra). Vector stocktiters ranged between 1-10 ×10¹² vg/mL.

Example 3 Efficient Gene Delivery Via the Mouse Vasculature Using AAVrh.74

With respect to clinical application, rather than deliveringalpha-sarcoglycan gene by direct injection into the muscle, meaningfulresults will be best attained using a gene transfer approach that hasthe ability to reach widespread muscle targets resulting in animprovement in the patient's quality of life. A vascular deliveryapproach allows for a one-time vector infusion to reach multiple musclesinstead of direct injections that would be necessary using a directinjection intramuscular approach. Moreover, benefits of a regionalvascular approach include: lack of widespread dissemination of virus;safe passage of the virus directly to the targeted muscles; andtransduction of multiple muscles in, for example, the leg.

AAVrh.74 Micro-Dystrophin Gene Delivery Versus AAV1 and AAV6 Delivery

The AAV1 serotype transduces muscle efficiently by direct intramuscularinjection, however comparative studies demonstrated that AAVrh.74delivered through the circulation is vastly superior to AAV1 andsuperior to AAV6 in transducing skeletal muscle via this route. Asdescribed in Rodino-Klapac et al., J. of Transl. Med, 5: 45 (2007), AAV6and AAV rh.74 carrying a micro-dystrophin gene demonstrated ease incrossing the vascular barrier when delivered to skeletal muscle in themdx mouse through a catheter in the femoral artery. Extremely efficientregional vascular delivery was observed using AAVrh.74.micro-dystrophin,and yielded percent transduced myofibers as follows: 94.5±0.9 (1 month),91.3±3.1 (2 months), and 89.6±1.6% (3 months). AAV6.micro-dystrophintreated animals demonstrated 87.7±6.8 (1 month), 78.9±7.4 (2 months),and 81.2±6.2% (3 months) transduction. In striking contrast, AAV1demonstrated very low transduction efficiency [0.9±0.3 (1 month),2.1±0.8 (2 months), and 2.1±0.7% (3 months)] by the vascular deliveryroute. The delivery of micro-dystrophin through the femoral artery wasaccompanied by functional improvement as measured by protection againstcontraction-induced injury and improvement in tetanic force.

AAVrh.74.tMCK.hSGCA Vascular Delivery in Knock-Out Mice

In the present experiments, the AAVrh.74.tMCK.hSGCA was delivered byisolated limb perfusion to the alpha-sarcoglycan knock-out mouse.

Sedated and anesthetized animals secured to a surgical platform wereprepared and draped in the usual sterile fashion. Suture-tourniquets(3.0 braided silk) were placed loosely around the thigh near theinguinal region. A small incision was placed over the femoral bundlevisible through the skin. The femoral artery was isolated and cannulatedwith a heat-pulled polyethylene (PE) 10 catheter prefilled with normalsaline and secured in place. The tourniquet was tightened and apre-flush of normal saline was delivered. Following the pre-flush, thevector dose 2×10¹²vg/kg wt was administered and allowed to dwell for 10minutes. After the 10-minute dwell a final post-flush of normal salinewas delivered, and the catheter and tourniquet removed and the animalrecovered.

Three-months post-gene transfer, transduction levels were observedaveraging 78.2±11% of muscle fibers. Not only was the transgeneappropriately expressed at the muscle fiber periphery in greater than75% of muscle fibers, muscle function (measured as specific force) wasrestored in treated animal muscles compared to non-treated muscle. Inother experiments, gene transfer of up to 90% positive fibers in thelower extremity musculature was observed.

Example 4

AAVrh.74.tMCK.hSGCA Vascular Delivery in Non-Human Primates

The above success in the mouse promulgated extensive studies innon-human primates using both cynomologus and rhesus macaques. In bothspecies, a clinically relevant, intra-arterial delivery system was used.

Sedated and anesthetized animals were secured to a surgical bed.Proximal and distal tourniquets were loosely positioned above the kneeand below the gastrocemius muscle of a macaque. A small incision wasplaced at the femoral triangle and the femoral artery was identified anddissected free and looped with proximal and distal ligatures to controlbleeding and facilitate catheter introduction. The femoral artery wascannulated with a 3.0 Fr introducer sheath via a modified Seldingermethod by passing the pre-flushed sheath over a wire previously placedin the artery. The sheath was advanced only a few centimeters andsecured in place with a 3.0 braided silk suture.

Heparinization was achieved with 50 U/kg body weight via the sheath andthe sheath was cleared with normal saline. Fluoroscopy was used togenerate a road map of the vasculature by administering a fewmilliliters of contrast agent through the sheath and capturing thefluoroscopic image. A 3.0 Fr, 50 cm long catheter was placed into theintroducer sheath and advanced a few centimeters. A guide wire (0.018in., diameter) was placed through the catheter and, under fluoroscopicguidance, advanced to the sural arteries, which perfuse the two heads ofthe gastrocnemius. Once the catheter was correctly positioned, thevascular bed of the gastrocnemius was isolated by the placement ofproximal and distal tourniquets. The proximal tourniquet was placedabove the knee and just proximal to the catheter tip. Optimal placementof the proximal tourniquet was assessed by partial tourniquet tighteningand visualization of a small volume (few milliliters) of injectedcontrast agent. Once the relationship of the proximal tourniquet tocatheter tip was established, the contrast was flushed from the limbwith normal saline and the distal tourniquet was positioned just belowthe gastrocnemius. The second tourniquet provides compartmentalizationof the gastrocnemius. Dosing began with a pre-flush volume (2.5 mL/kg)of normal saline delivered over 60 sec. with the tourniquets pulledsnug. While the final volume was administered, the tourniquets werepulled tight to occlude blood flow. With the tourniquets pulled tightthe rAAV vector carrying the gene of interest, AAVrh.74.tMCK.hSGCA(2×10¹² viral genomes per kg in 2.5 mL per kg volume), was administeredover 60 s. Allow 10 min. dwell time with the tourniquets left tight.Following the 10 min dwell and with the tourniquets still tight andoccluding blood flow, a post-volume of normal saline (2.5 mL/kg) wasadministered over 60 s. At the completion of dosing the tourniquets andcatheter were removed and direct pressure was applied to the wound for10 min to control bleeding. The wound was closed with a continuoussubcuticular 4.0 Vicryl suture. Apressure dressing was applied to thesite and kept in place until the animal awoke from anesthesia.

Following the above vector delivery protocol, similarly treated animalswere sacrificed 12 to 24 weeks later and muscle samples were removed forstorage and study. Gene expression was measured by antibody staining ofthe transgene expression product in situ.

Muscle transduction exceeded 75% in the muscles of interest using dosesapplicable to a clinical trial. Evaluation of antibody stainedmicroscopic images of the treated muscles showed that micro-dystrophin,alpha-sarcoglycan or a FLAG-tag (6 amino acid tag attached to thetransgene) was expressed at the fiber periphery, the region known as thesarcolemma. This is the region of normal expression for these proteins.Muscles not targeted had very low levels of transgene expressionhighlighting the specific nature of the targeting. Robust expression inother animals treated was observed for up to six months.

Example 5 AAVrh.74 Vector Biodistribution

By using the femoral artery delivery approach described in Example 4,vector escape outside the limb was minimized as shown by PCR-baseddetection of AAV vector genomes in organs throughout the body at thetime of animal necropsy. FIG. 6 shows vector biodistribution data fromfifteen monkeys receiving vector through the femoral artery. Only thetargeted muscle (gastrocnemius) and spleen shows significant number ofvector genomes. These samples were obtained three weeks post-genedelivery through the femoral artery. The number of vector genomesrecovered from remote sites were negligible (note the log scale).

Example 6 Dose Escalation Study

A dose escalation study of AAVrh74.tMCK.hSGCA delivered via the femoralartery to the quadriceps muscles of both legs of LGMD2D(alpha-sarcoglycan-deficient) patients is performed. Two cohorts undergogene transfer in a standard three-six dose escalation scheme toestablish maximum tolerated dose (MTD) using toxicity. A minimum ofthree subjects are enrolled into each cohort. The first cohort receivesa total dose of 3×10¹³ vg split between the two extremities (1.5×10¹³ vgper limb). The vector is infused through the femoral artery using apercutaneous balloon catheter. This is a one-time vector infusion. Thesecond cohort receives 1×10¹⁴ vg total dose - split between the twoquads (5×10¹³ vg per limb) delivered to the quadriceps muscles accordingto the same protocol. All patients undergo a muscle biopsy at 3 months(one leg), and 6 months (contra lateral leg) post-gene therapy.

More specifically, patients receive general anesthesia during theprocedure. Procedures are performed under sterile conditions. Thefemoral arteries are catheterized percutaneously in the groin. Afluoroscopy guided 5 Fr catheter is advanced to the vessels supplyingthe quadriceps muscle. A blood pressure cuff at the knee serves as atourniquet to promote vector delivery to the quadriceps muscles. Aballoon catheter prevents backflow of vector to general circulation.Blood flow to the extremity is occluded for 10 minutes to promotetransport through the endovascular barrier. Prior to vectoradministration, a pre-vector flush of saline (2.5 ml/kg) is given overone minute, immediately followed by occluding blood flow to theextremity. AAVrh.74.tMCK.hSGCA is infused over 60 seconds at a dose of1.5×10¹³ vg per limb in 2.5 ml/kg of Tris buffered saline for thelow-dose cohort, and 5×10¹³ vg per limb in 2.5 ml/kg of Tris bufferedsaline for the higher dose cohort. The extremity remains isolated fromthe circulation for 10 minutes before releasing the tourniquet. Apost-vector flush (2.5 ml/kg) is infused over one minute prior torelease of tourniquets. Direct pressure is applied for 10 minutes toensure hemostasis.

Patients undergo muscle biopsies at two time points, three and sixmonths (on contralateral limbs). Biopsy evaluation includes analysis ofalpha-sarcoglycan expression and the entire sarcoglycan complex byimmune stains and western blots. Mononuclear cells (CD4+and CD8+,macrophages) are assessed as is MHC I and II expression. On a monthlybasis, patents are evaluated for neutralizing antibodies to rAAV8 alongwith ELISpots to both rh.74 capsid and alpha-sarcoglycan protein. Musclestrength of the quadriceps is evaluated by quantitative myometry andtimed functional tests of standing from a sitting position and walking 9meters.

Example 7 Isolated Whole Limb Re-Circulation (IWLRC) Protocol

Some chemotherapeutic agents have been delivered by limb perfusion asdescribed in Justison et al., JECT, 41: 231-234 (2009) and van Akkooi etal., Eur. J. Cardio-thoracic Surgery, 30: 408-410 (2006). It iscontemplated herein that recombinant viruses of the invention can alsobe delivered to a patient via a re-circulating methodology. Themethodology provides controlled dwell time for viral uptake, control ofperfusion pressure, vascular pH, vascular oxygenation and clearing ofplasma/blood containing antibodies and complement from the targetedcirculation and tissue. In brief, a limb of a patient is isolated with atourniquet, an artery and vein of the limb are accessed withangio-catheters and the two catheters are connected via tubing,stopcocks and a pump. Buffered solution is pumped into the artery andblood and serum is collected from the limb into a sterile bag forredelivery upon completion of the procedure. While the limb is perfusedwith buffered solution, the viral vector is administered.

More specifically, to deliver AAVrh.74.tMCK.hSGCA to a lower limb of apatient for example, the patient is sedated and anesthetized. Theinguinal area is prepared and draped in the usual sterile fashion.Appropriately sized angio-catheters are placed via direct cut down andblunt dissection into the femoral artery and vein at a site just distalto the inguinal ligament allowing enough space to place a tourniquet.The tourniquet allows temporary isolation of the lower extremity.Alternatively, it is contemplated that angio-catheters can be placedpercutaneously or at distal sites and targeted by fluoroscopy.

To these angio-catheters a sterile 3/16″ (ID) venous line is connectedto the venous catheter with a luer lock. The tubing will contain two3/16″ single luer connectors separated by a three-inch piece of 3/16″tubing. Each 3/16″ luer connector will have an associated six-inchpigtail and two-way stopcock. This allows for collection of the blood asit is displaced with a Normosol-R (Hospira Inc., Lake Forest, Ill.)solution. The blood will be mixed with 8 ml ACD-A anticoagulant duringcollection so that imay be returned post-procedure. From the second3/16″ double luer connector is again be 3/16″ tubing that is placedwithin one of the roller-heads of a Maqet HL-20 twin roller pump(Maquet, Hirrlingen, Germany). This roller-head serves as the perfusatepump during the experiment. Post roller-head the 3/16″ tubing isconnected to a Sorin CSC 14 heat exchanger (Sorin Group USA, Inc.,Arvada, Colo.) (28 ml prime volume). The CSC 14 allows for temperatureregulation of the perfusate throughout the procedure. A two-inch pieceof 3/16″ tubing is connected to the outlet of the CSC 14 heat exchangerwhere a 3/16″ single luer connector and associated six-inch pigtail andstopcock are connected. A two-inch piece of 3/16″ tubing is connected tothe opposite end of the 3/16″ single luer and is then stepped down to ⅛″(ID) tubing that serves as the return line. The return line is connectedto the catheter within the artery with a luer connection. All componentsare primed with Normosol-R in a sterile manner, and warmed to 37 degreesCelsius prior to connection with the arterial and venous catheters byrecirculating through a bag of Normosol-R. The total prime volume of allcomponents is 62 mL+/−10 mL.

Once connected to the venous and arterial cannulas, a tubing clamp isplaced between the two 3/16″ luer connectors on the venous limb.Normosol-R is injected into the distal luer connector utilizing a 60 mLsyringe, displacing the blood into a 60 mL syringe (containing 8 mLACD-A) attached to the proximal 3/16″ luer. This process is repeateduntil the drainage (blood+Normosol) have an immeasurable hematocrit (<6g/dL). The tubing clamp is removed and limb perfusion withAAVrh.74.tMCK.hSGCA begins. During limb perfusion, venous (drain)pressure is monitored utilizing a disposable pressure transducerconnected to the HL-20 pump and to one of the 3/16″ luer connectorswithin the venous line. The pressure is not allowed to be less than −50mmHg. To insure the pressure does not go more negative than this, servoregulation of the pump is set to −50 mmHg. As the pressure approachesthis pressure, the roller-head automatically slows or stops preventingdamage to the vessel. Arterial (return) pressure monitoring is completedin the same manner on the 3/16″ luer connector on the return line. Thisservo regulation is set to 200 mmHg. The perfusion flow rate is set at50 mL/min and maintained for one hour.

At the conclusion of one hour of re-circulation, the blood andNormosol-R initially withdrawn during the connection process will bereturned. To achieve this, a tubing clamp is placed between the two3/16″ luer connectors on the venous limb. An empty 60 mL syringe isconnected to the proximal luer connector. The 60 mL syringes collectedearlier are connected and injected via the distal luer connector inreverse order of their collection. Once the blood has been returned, thecircuit is disconnected from the luer connectors and disposed of asbiohazard waste. The tourniquet is removed slowly to allow systemiccirculation to the limb and the cannulae removed. Pressure is used tocontrol bleeding at the cut down site. Following the procedure, thepatient is recovered in an appropriately warmed environment.

Example 8 IWLRC in the Non-Human Primate with a Reporter Construct

IWLRC with AAVrh.74 and a reporter transgene construct comprising acytomegalovirus promoter and eGFP (AAVrh.74.CMV.eGFP) demonstratesefficiently expressed transgene with broad distribution throughout themajor muscles of the lower limb.

Two vector/transgene doses, high 6×10¹² vg/kg and low 2×10¹² vg/kg, wereadministered to the lower extremities of two rhesus macaques, such thatone animal received the low dose to both lower limbs and the otheranimal received the high dose to both lower limbs. Results achieved withthe doses are presented in FIGS. 7 and 8, respectively.

On analysis of the major muscles of the lower extremity, both doses showbroad transgene expression throughout the lower extremity with broaderand more efficient expression in the lower extremity of the higher dosedanimal. At the dose of 6×10¹², IWLRC resulted in greater than 40% musclefiber transgene expression in major muscles of the lower extremityexcept the biceps femoris (HBF) and gracilis (HGras) of the Hamstringmuscle group. Included in the graphs but not specifically targeted inthis protocol as part of the lower limb are the Gluteus (max and med)muscles; broad expression in the gluteus medius and less in the gluteusmaximus is noted.

Example 9

IWLRC in the Non-Human Primate with Therapeutic Transgenes

IWLRC was performed in non-human primates using AAVrh.74 to deliver atherapeutic micro-dystrophin transgene or a therapeuticalpha-sarcoglycan transgene (specifically using AAVrh.74.tMCK.hSGCA).Results achieved with the transgenes are presented in FIGS. 9 and 10,respectively.

The transgenes were expressed with broad distribution throughout themajor muscles of the lower limb.

Example 10 Vascular Delivery of AAVrh.74.tMCK.hSGCA to Alpha-SarcoglycanKnock-Out Mice

A two-dose escalation study was performed in alpha-sarcoglycan knock-outmice. The two doses were 6×10¹¹ vg/kg (low) and 2×10¹²vg/kg (high). Thefemoral artery of mice was catheterized and AAV74.tMCK.hSGCA wasdelivered at high or low dose in 100 μl. A tourniquet placed mid-thighcontained vector delivery to the lower extremity, limiting delivery tothe lower limb muscles. Three months post-gene transfer, lower limbmuscles were harvested and assessed for resistance to eccentriccontraction based injury and tetanic force.

Efficacy was demonstrated at both high and low dose. There wassignificant improvement versus alpha-sarcoglycan knock-out controls atboth high and low dose. The high dose was not significantly differentthan wild-type mice (ANOVA). See FIG. 11.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the claims should be placed on the invention.

All documents referred to in this application are hereby incorporated byreference in their entirety.

We claim:
 1. A method of ameliorating limb girdle muscular dystrophytype 2D (LGMD) in a patient in need thereof comprising the step ofperfusing the vasculature of a limb of the patient with a recombinantadeno-associated virus comprising the AAV rh.74 capsid of SEQ ID NO: 2and comprising the alpha-sarcoglycan polynucleotide of SEQ ID NO: 3 in agene expression cassette in the virus genome.
 2. A method of inhibitingthe progression of dystrophic pathology associated with LGMD 2D in apatient in need thereof comprising the step of perfusing the vasculatureof a limb of the patient with a recombinant adeno-associated viruscomprising AAV rh.74 capsid of SEQ ID NO: 2 and comprising thealpha-sarcoglycan polynucleotide of SEQ ID NO: 3 in a gene expressioncassette in the virus genome.
 3. A method of improving muscle functionin a patient afflicted with limb girdle muscular dystrophy type 2D (LGMD2D) comprising the step of perfusing the vasculature of a limb of thepatient with a recombinant adeno-associated virus comprising AAV rh.74capsid of SEQ ID NO: 2 and comprising the alpha-sarcoglycanpolynucleotide of SEQ ID NO: 3 in a gene expression cassette in thevirus genome.
 4. The method of claim 3 wherein the improvement in musclefunction is an improvement in muscle strength.
 5. The method of claim 3wherein the improvement in muscle function is an improvement instability in standing and walking.
 6. The method of any of claims 1-5wherein the virus genome is a self-complementary genome.
 7. The methodof any of claims 1-6 wherein the recombinant adeno-associated virus isAAVrh.74.tMC K.hSGCA.
 8. A method of delivering an alpha-sarcoglycanpolynucleotide to an animal in need thereof, comprising the step ofperfusing the vasculature of a limb of the animal with a WO 2013/078316PCT/US2012/066265 recombinant adeno-associated virus comprising the AAVrh.74 capsid of SEQ ID NO: 2 and comprising the alpha-sarcoglycanpolynucleotide of SEQ ID NO: 3 in a gene expression cassette in thevirus genome.
 9. The method of claim 8 wherein genome of the rAAV lacksAAV rep and cap DNA.
 10. The method of claim 9 wherein the virus genomeis a self-complementary genome.
 11. The method of claim 9, 10 or 11wherein the recombinant adeno-associated virus is AAVrh.74.tMC K.hSGCA.12. A recombinant adeno-associated virus (AAV) comprising the AAV rh.74capsid of SEQ ID NO: 2 and comprising the alpha-sarcoglycanpolynucleotide of SEQ ID NO: 3 in a gene expression cassette in thevirus genome.
 13. The rAAV of claim 12 wherein genome of the rAAV lacksAAV rep and cap DNA.
 14. The rAAV of claim 12 or 13 wherein the genomeis a self-complementary genome.
 15. The rAAV of any of claims 12-14wherein the rAAV is AAVrh.74.tMCK.hSGCA.
 16. A method of delivering arecombinant adeno-associated virus comprising: connecting arecirculation circuit to a vasculature of a limb; isolating circulationof the limb from the remainder of the body; adding a recombinantadeno-associated virus comprising the AAV rh.74 capsid of SEQ ID NO: 2and comprising the alpha-sarcoglycan polynucleotide of SEQ ID NO: 3 in agene expression cassette in the virus genome to the recirculationcircuit; passing the recombinant adeno-associated virus through therecirculation circuit and the limb repeatedly; and disconnecting therecirculation circuit from the limb.
 17. The method of claim 16, furthercomprising: removing a volume of blood from the limb after isolating thelimb from the remainder of the body; introducing a non-blood perfusateto the recirculation circuit and the limb prior to adding therecombinant adeno-associated virus to the recirculation circuit; andpassing the non-blood perfusate through the recirculation circuit andthe limb repeatedly as the recombinant adeno-associated virus is passedthrough the recirculation circuit and the limb.
 18. The method of claim17, wherein the non-blood perfusate comprises a buffer solution.
 19. Themethod of claim 17, further comprising: removing the non-blood perfusateafter repeatedly passing the recombinant adeno-associated virus and thenon-blood perfusate through the recirculation circuit and the limb; andreintroducing the volume of blood to the limb after removing thenon-blood perfustate.
 20. The method of any one of claim 16, 17, 18 or19, wherein the recombinant adeno-associated virus is passed through therecirculation circuit and the limb for at least 30 minutes.
 21. Themethod of any one of claim 16, 17, 18, 19 or 20 wherein genome of therAAV lacks AAV rep and cap DNA.
 22. The method of any one of claim 16,17, 18, 19, 20 or 21 wherein the virus genome is a self-complementarygenome.
 23. The method of any one of claim 16, 17, 18, 19, 20, 21 or 22wherein the recombinant adeno-associated virus is AAVrh.74.tMCK.hSGCA.